Published in Volume
117, Issue 6
(June 1, 2007)J Clin Invest.
Copyright © 2007, American Society for Clinical
Vascular biology and bone formation: hints from HIF
Department of Medicine, Center for Cardiovascular Research, Division of Bone and
Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri,
Address correspondence to: Dwight A. Towler, Washington University School
of Medicine, Barnes-Jewish North Campus Box 8301, 660 South Euclid Avenue, St.
Louis, Missouri 63110, USA. Phone: (314) 454-7434; Fax: (314) 454-8434; E-mail:
Published June 1, 2007
In this issue of the JCI, Wang, Clemens, and colleagues
demonstrate that hypoxia-inducible factor α (HIFα)
signaling in bone-building osteoblasts is central to the coupling of
angiogenesis and long bone development in mice (see the related article
beginning on page 1616). They show that bone formation controlled by osteoblast
HIFα signaling is not cell autonomous but is coupled to skeletal
angiogenesis dependent upon VEGF signaling. Thus, strategies that promote
HIFα signaling in osteoblasts may augment bone formation and
accelerate fracture repair.
Tremendous unmet clinical needs exist in musculoskeletal medicine. The direct costs of
musculoskeletal diseases in the Unites States are well over $100 billion per annum
(1). Osteoporosis and osteoarthritis are
recognized as common and clinically important, but other serious skeletal diseases also
afflict our populace. In the setting of type 2 diabetes mellitus (T2DM), lower-extremity
musculoskeletal disease is prevalent, costly, and exceedingly difficult to manage, with
fracture, arthropathy, ischemia, ulcer, and infection commonly confronting patients and
clinicians. The total costs associated with lower-extremity amputation in T2DM alone are
greater than the combined costs of treating fatal and nonfatal
myocardial infarction associated with T2DM (2).
Stage-specific and disease-specific strategies are necessary to safely promote bone
formation in individuals with: (a) underlying vasculopathies, such as those associated
with diabetes or renal failure; (b) underlying malignancy of any sort; (c) extant
osteoporosis that has removed trabecular templates for bone apposition; (d) osteoporosis
in the setting of childhood growth and open epiphyses; and (e) drug- or
coagulopathy-related disorders that cause avascular necrosis.
In this issue of the JCI, the study by Wang et al. (3) affords us a better understanding of the mechanisms
coupling bone and vascular physiology, providing insights useful for devising novel
strategies to address the mounting unmet needs in orthopedic medicine. The vasculature
provides: (a) a sustentacular niche and source of adult mesenchymal stem cells,
including osteoprogenitors; (b) the organizational and rate-limiting “point
of reference” for Haversian bone formation; and (c) the conduit for calcium,
phosphate, hematopoietic, and nutrient supply necessary for mineralization and calcium
mobilization (4–11). We know precious little about how the vasculature
integrates and conveys signals during skeletogenesis. However, VEGF (9) has emerged as the prototypic osteogenic-angiogenic
coupling factor (10). The VEGF gene encodes a
secreted polypeptide globally required for vasculogenesis and angiogenesis (11). Bioactivity is modulated by VEGF gene
expression — and differential splicing that generates three unique gene
products — with signals transduced via specific VEGF receptor tyrosine
kinases (9). The name
“VEGF” belies its contributions to osteoblast ontogeny,
chondrocyte physiology, and osteoclast formation (10). VEGF signaling is tightly regulated in bone, coupled to morphogenetic,
metabolic, inflammatory, and mechanical cues that control mineral metabolism. In
addition to regulating the expansion and survival of mesenchymal progenitors (Figure
1 and below), signals provided by VEGFR2
mediate angiogenic cross-talk with TNF receptor 2 (TNFR2) (12), critical for postnatal defense against limb ischemia
(13). Given the contributions of
bone-vascular interactions to all aspects of bone biology, our limited understanding of
this important physiology impedes development of novel bone anabolic therapies.
Working model of osteogenic-angiogenic coupling in trabecular bone. Recent data from multiple laboratories (16–24) have
indicated that microvascular smooth muscle cells known as pericytes
represent osteoprogenitors capable of bone formation when placed in the
correct microenvironment. Pericytes appear to arise from a vessel-associated
stem cell progenitor (mesoangioblast; refs. 20, 31, 32), and during the process of mesoderm growth
and angiogenesis, this VEGFR2-expressing stem cell undergoes expansion
(23, 31). In this issue of the JCI,
Wang et al. (3) demonstrate that
osteoblast HIFα subunits, transcriptional regulators of
VEGF expression, represent rate-limiting components of
osteogenic-angiogenic coupling and trabecular bone formation. Augmentation
of osteoblast HIFα expression and bone formation was achieved by
conditionally deleting Vhl, the gene encoding pVHL
— the E3 ubiquitin ligase necessary for HIFα
degradation. Bone formation was not cell autonomous — i.e., not
dependent solely on osteoblast functions — but required
VEGF-mediated paracrine signals in bone that stimulated angiogenesis. Since
VEGF can expand VEGFR2-expressing mesoangioblast numbers during angiogenesis
(33), this process may drive the
increase in osteoblast numbers that promotes massive trabecular bone
formation in the osteogenic marrow environment. PHD enzyme activity is also
required for HIFα degradation, oxidatively
“tagging” HIFα for recognition by pVHL.
In addition to low oxygen levels (as shown here), mechanical stimuli,
TNF-α, and reactive oxygen species can also upregulate
HIFα expression (29).
Strategies that augment osteoblast HIFα/VEGF signaling by
selectively inhibiting skeletal PHD may increase bone formation and enhance
fracture healing. BMSC, bone marrow stromal cell; CVC, calcifying vascular
cell; GTF, general transcription factor; Pol II, RNA polymerase II; Ub,
New hints from HIF
In their current study, Wang, Clemens, and colleagues (3) significantly advance our understanding of bone-vascular coupling by
establishing the critical role for osteoblast hypoxia-inducible factor
1α (HIF-1α) and HIF-2α in bone formation (Figure
1). As recently reviewed (14), HIFs are components of heterodimeric,
hypoxia-activated transcription factor complexes that bind to well-characterized DNA
cognates called hypoxia-response elements, activating the expression of genes such
as VEGF and erythropoietin that can improve tissue
oxygen delivery (15). Dependent upon cellular
context, HIF-1α and HIF-2α may or may not exhibit functional
redundancy (14). The Clemens group first
showed that osteoblasts express all necessary components of the oxygen-sensing
pathway in addition to HIF-1α and HIF-2α (herein
collectively referred to as HIFα). These components include the
oxygen-dependent prolyl hydroxylases (PHDs) that “tag”
HIFα for recognition by the E3 ubiquitin ligase von
Hippel–Lindau protein (pVHL) and pVHL itself, necessary for
HIFα proteosomal degradation (3)
(Figure 1). Moreover, the authors demonstrate
hypoxia-dependent nuclear accumulation of HIFα and concomitant
upregulation of VEGF expression, indicating intact oxygen-sensing
functions in osteoblasts. To evaluate the biological importance of osteoblast HIF
signaling in vivo, the authors used Cre-lox technology; they implemented the
bone-specific human osteocalcin promoter as a delivery module for Cre recombinase
expression in mice possessing floxed Vhl alleles. This strategy
abrogates pVHL accumulation in mature osteoblasts (3). In this conditional knockout, denoted Δ
Vhl, osteoblast accumulation of both HIF-1α and
HIF-2α was induced due to the absence of pVHL-dependent degradation
(Figure 1). The in vivo effects on long bone
formation were remarkable; bone volume/tissue volume (BV/TV) increased by 70% with
osteoblast-specific induction of HIFα subunits. Detailed
histomorphometry revealed very early postnatal increases in trabecular osteoblast
numbers during long bone modeling — numbers that
“normalized” once a new steady state of high bone mass was
achieved in Δ Vhl mice (3). In culture, osteoblasts possessing floxed Vhl
alleles also upregulated HIFα when transduced with an adenoviral Cre
vector — with concomitant induction of VEGF.
HIFα induction did not alter cultured osteoblast proliferation rate,
apoptotic rate, matrix synthetic activity, or mineral deposition ex vivo; thus, the
bone anabolic actions of osteoblast HIFα induction observed in vivo were
not cell autonomous. Bone histomorphometry and serum biochemistries showed no
decrements in bone-resorbing osteoclast numbers or activity, excluding overt
contributions of osteoclast insufficiency to increased BV/TV in Δ
Vhl mice. However, a profound increase in bone vascularity was
observed for the Δ Vhl mice (3). Moreover, a massive increase in capillary sprouting
was exhibited by Δ Vhllong bones maintained in organ
culture (3). Angiogenic responses were
dependent upon paracrine VEGF actions, since: (a) VEGF-neutralizing antibody
abrogated sprouting; and (b) skeletal production of VEGF mRNA was
increased in Δ Vhl mice without changes in circulating
VEGF. Relationships to HIFα-dependent angiogenesis and bone formation
were further confirmed using mice in which Hif1a was conditionally
knocked out in osteoblasts (Δ Hif1a mice). Bone volume
and vascularity were reduced in Δ Hif1amice, with
reductions partially offset by compensatory HIF-2α expression. The
overlapping redundancy of the latter was diligently demonstrated using a third mouse
model, Vhlgene deletion in Δ Hif1a
mice; these Δ Vhl/Δ Hif1a
mice exhibited markedly increased HIF-2α protein accumulation, with
concomitant restoration of bone volume and angiogenesis (3). Thus, the authors conclude that osteoblast
HIFα signaling is a central component of rate-limiting,
osteogenic-angiogenic coupling that controls long bone formation. This coupling
occurs via mechanisms that are not cell autonomous and that utilize paracrine VEGF
angiogenic signals to expand osteoblast numbers during long bone development (3).
Angiogenesis and osteoblast ontogeny
How, then, might the angiogenic response and osteogenic-angiogenic coupling increase
osteoblast numbers and provide the massive marrow bone formation observed (3)? Recently, several groups have identified the
microvascular smooth muscle cell, the pericyte, as an important osteoprogenitor
(16–24) (Figure 1).
Demer, Canfield, and colleagues have shown that the pericyte exhibits
multipotentiality, capable of osteogenic, chondrogenic, adipogenic, and SMC
differentiation (17, 18). Molecularly, pericytes express early features of the
VSMC lineage, including smooth muscle 22 kDa (SM22), α-SMC, and
species-specific gangliosides demarcated by the 3G5 monoclonal antibody (17, 19).
Anatomically, the pericyte is intimately juxtaposed to the endothelial capillary
network. In the marrow microenvironment, the bone marrow stromal cell exhibits the
histoanatomic characteristics of the pericyte (16). Thus, from this perspective the bone marrow stromal cell —
the osteoprogenitor — can be viewed as a tissue-specific pericyte.
What is the ontogeny of the vascular pericyte? Cossu and colleagues provide data
suggesting that the mesoangioblast (20), a
vessel-associated mesenchymal stem cell with the capacity to differentiate into
cells of endothelial and VSMC lineages, might be the source of pericytes. Studies of
differentiating murine ES cells confirm the existence of a highly plastic,
VEGFR2+ endothelial-SMC progenitor — a mesenchymal stem
cell that can give rise to mineralizing osteoblasts in culture via pericytic SMC
lineage (23). Skeletal osteoblasts can and do
arise from the pericyte cell lineage in vivo; SM22-positive and
α-SMC–positive cells “coregister”
with preosteoblasts identified using Col3.6-GFP reporter mice following induction of
de novo osteogenesis (24). Thus, concomitant
with angiogenic sprouting, VEGF/VEGFR2 signaling is posited to expand the potential
osteoprogenitor pool via the pericyte intermediate (Figure 1). However, until better pericyte lineage markers are
developed, or the effects of HIFα expansion are tested in the lineage
reporter mice (24), the mechanisms proposed
Location, location, location
Remarkably, unlike in long bone, little if any in vivo effect of osteoblast
Vhl deletion was observed by Wang et al. in calvarial bone
(3). Why might this occur? Unique and
differentially regulated ontogeny and angiogenic responses likely contribute (25). There are places in the skeleton (e.g.,
neural crest–derived calvarial bone, lateral components of the clavicle,
the mid-diaphyseal collar of long bone) where bone forms via nonendochondral
mechanisms (11, 25). Denoted as intramembranous ossification, this
osteoblast-mediated mineral deposition occurs directly in the type I
collagen–based extracellular matrix — without replacement of
a precedent, avascular cartilaginous template by bone and marrow as is required for
endochondral ossification (10, 25). In the developing skull, it is probable
that cranial suture and dural mechanical tension organize angiogenesis necessary for
intramembranous ossification (26–28). VEGF
expression in osteoblasts is mechanically very responsive (29). Distraction osteogenesis — an orthopedic
mechanical manipulation that promotes robust angiogenesis and bone formation via
nonendochondral mechanisms — upregulates both HIF-1α and
VEGF (29). Thus, it is tempting to speculate
that the differences observed by Wang et al. (3) arise due to differences in the rate-limiting stimuli that control
osteogenic-angiogenic coupling in calvarial versus long bone development. For the
moment, however, the precise reasons for the differences observed between long bone
and calvarial bone formation following Vhl deletion remain to be
Many questions remain to be answered. Although HIFα clearly regulates
osteogenic-angiogenic interactions necessary for bone formation, other secreted
molecules in addition to VEGF, such TNF-α (6, 12, 13) and FGF2 (5, 27, 28), might contribute to this coupling. Potential
contributions of paracrine TNF-α signaling — an important
activator of TNFR2-VEGFR2 cross-talk (12,
13) and bone formation (6) — have yet to be detailed. Should
administration of bevacizumab, a clinically useful inhibitory antibody to VEGF
(30), be shown to abrogate the bone
anabolic effects of osteoblast HIFα, this would provide pharmacologic
evidence that paracrine VEGF signals are nonredundant in osteogenic-angiogenic
coupling. Moreover, since VEGF induces the production of bone morphogenetic protein
(BMP) by endothelial cells (5), the
consequences of inactivating endothelial BMP expression would help support the
evolving working model (Figure 1). The
mechanisms that punctuate feed-forward osteogenic-angiogenic coupling are not known
but clearly exist, since osteoblast numbers and bone formation quickly normalizes
postnatally at a higher bone mass (3). The
“osteostat” mechanism responsible for this physiologic
response will be extremely important to delineate — and may be
metabolically as well as mechanically determined. It may be possible to selectively
augment skeletal HIF-1α action — potentially by inhibiting
specific PHDs — as one strategy to promote bone formation and fracture
healing. It will be important to evaluate how the material and geometric properties
of bone manipulated via the HIFα pathway impact bone strength. Finally,
better markers are required to unambiguously characterize precursor-product
relationships in the mesoangioblast/pericyte/osteoblast lineage; such ontogeny is
likely to contribute to changes in trabecular bone mass dependent upon osteoblast
HIFα signaling (16, 18). All in all, novel and very important
biological principles emerge from the current study (3), i.e., that osteogenesis and angiogenesis are functionally coupled in
the marrow microenvironment by osteoblast HIFα signaling. Thus, in
addition to osteoblast and osteoclast lineages, the contributions of endothelial
cell precursors and their progeny (Figure 1)
must be considered in robust studies of bone formation and skeletal homeostasis.
The author is supported by NIH grants HL69229, HL81138, AR43731, and the
Barnes-Jewish Hospital Foundation.
Nonstandard abbreviations used: BMP, bone morphogenetic protein;
BV/TV, bone volume/tissue volume; HIF, hypoxia-inducible factor;
ΔHif1a mice, mice with
conditional deletion of Hif1a in osteoblasts; PHD, prolyl
hydroxylase; pVHL, von Hippel–Lindau protein; SM22, smooth muscle 22
kDa; T2DM, type 2 diabetes mellitus; TNFR2, TNF receptor 2;
ΔVhl mice, mice with conditional deletion of
Vhl in osteoblasts;
mice with conditional deletion of both Vhl and
Hif1a in osteoblasts.
Conflict of interest: The author receives grant support from the
NIH and the Barnes-Jewish Hospital Foundation and receives compensation as an ad
hoc consultant for Novartis, Lilly, and GlaxoSmithKline.
Citation for this article:J. Clin. Invest.117:1477–1480 (2007). doi:10.1172/JCI32518.
See the related article beginning on page 1616.
Woolf, A.D., Pflefer, B. 2003. Burden of major musculoskeletal conditions. Bull. World Health Organ. 81:646-656.
Clarke, P., Gray, A., Legood, R., Briggs, A., Holman, R. 2003. The impact of diabetes-related complications on healthcare costs:
results from the United Kingdom Prospective Diabetes Study (UKPDS Study No.
65). Diabet. Med. 20:442-450.
Wang, Y., et al. 2007. The hypoxia-inducible factor α pathway couples
angiogenesis to osteogenesis during skeletal development. J. Clin. Invest. 117:1616-1626.
Eghbali-Fatourechi, G.Z., et al. 2005. Circulating osteoblast-lineage cells in humans. N. Engl. J. Med. 352:1959-1966.
Bouletreau, P.J., et al. 2002. Hypoxia and VEGF up-regulate BMP-2 mRNA and protein expression in
microvascular endothelial cells: implications for fracture healing. Plast. Reconstr. Surg. 109:2384-2397.
Gerstenfeld, L.C., et al. 2003. Impaired fracture healing in the absence of TNF-alpha signaling:
the role of TNF-alpha in endochondral cartilage resorption. J. Bone Miner. Res. 18:1584-1592.
Sorescu, G.P., et al. 2004. Bone morphogenic protein 4 produced in endothelial cells by
oscillatory shear stress induces monocyte adhesion by stimulating reactive
oxygen species production from a nox1-based NADPH oxidase. Circ. Res. 95:773-779.
Csiszar, A., et al. 2006. Bone morphogenetic protein-2 induces proinflammatory endothelial
phenotype. Am. J. Pathol. 168:629-638.
Ferrara, N. 2004. Vascular endothelial growth factor: basic science and clinical
progress. Endocr. Rev. 25:581-611.
Zelzer, E., Olsen, B.R. 2005. Multiple roles of vascular endothelial growth factor (VEGF) in
skeletal development, growth, and repair. Curr. Top. Dev. Biol. 65:169-187.
Zelzer, E., et al. 2002. Skeletal defects in VEGF(120/120) mice reveal multiple roles for
VEGF in skeletogenesis. Development. 129:1893-1904.
He, Y., et al. 2006. Critical function of Bmx/Etk in ischemia-mediated arteriogenesis
and angiogenesis. J. Clin. Invest. 116:2344-2355.
Goukassian, D.A., et al. 2007. Tumor necrosis factor-alpha receptor p75 is required in
ischemia-induced neovascularization. Circulation. 115:752-762.
Ratcliffe, P.J. 2007. HIF-1 and HIF-2: working alone or together in hypoxia? J. Clin. Invest. 117:862-865.
Forsythe, J.A., et al. 1996. Activation of vascular endothelial growth factor gene
transcription by hypoxia-inducible factor 1. Mol. Cell. Biol. 16:4604-4613.
Shi, S., Gronthos, S. 2003. Perivascular niche of postnatal mesenchymal stem cells in human
bone marrow and dental pulp. J. Bone Miner. Res. 18:696-704.
Farrington-Rock, C., et al. 2004. Chondrogenic and adipogenic potential of microvascular pericytes. Circulation. 110:2226-2232.
Tintut, Y., et al. 2003. Multilineage potential of cells from the artery wall. Circulation. 108:2505-2510.
Schor, A.M., Allen, T.D., Canfield, A.E., Sloan, P., Schor, S.L. 1990. Pericytes derived from the retinal microvasculature undergo
calcification in vitro. J. Cell Sci. 97:449-461.
Brunelli, S., et al. 2004. Msx2 and necdin combined activities are required for smooth
muscle differentiation in mesoangioblast stem cells. Circ. Res. 94:1571-1578.
Shao, J.S., et al. 2005. Msx2 promotes cardiovascular calcification by activating
paracrine Wnt signals. J. Clin. Invest. 115:1210-1220.
Cheng, S.L., Shao, J.S., Charlton-Kachigian, N., Loewy, A.P., Towler, D.A. 2003. MSX2 promotes osteogenesis and suppresses adipogenic
differentiation of multipotent mesenchymal progenitors. J. Biol. Chem. 278:45969-45977.
Sakurai, H., et al. 2006. In vitro modeling of paraxial and lateral mesoderm
differentiation reveals early reversibility. Stem Cells. 24:575-586.
Kalajzic, I., et al. 2006. Myofibroblast/pericyte phenotype of the osteoprogenitor cell. J. Bone Miner. Res. 21: S2 , abstract
Eames, B.F., de la Fuente, L., Helms, J.A. 2003. Molecular ontogeny of the skeleton. Birth Defects Res. C Embryo Today. 69:93-101.
Burrows, A.M., et al. 2001. Endocranial vascular patterns in a familial rabbit model of
coronal suture synostosis. Cleft Palate Craniofac. J. 38:615-621.
Henderson, J.H., Longaker, M.T., Carter, D.R. 2004. Sutural bone deposition rate and strain magnitude during cranial
development. Bone. 34:271-280.
Fong, K.D., et al. 2003. Mechanical strain affects dura mater biological processes:
implications for immature calvarial healing. Plast. Reconstr. Surg. 112:1312-1327.
Carvalho, R.S., et al. 2004. The role of angiogenesis in a murine tibial model of distraction
osteogenesis. Bone. 34:849-861.
George, D.J., Kaelin (Jr.), W.G. 2003. The von Hippel-Lindau protein, vascular endothelial growth
factor, and kidney cancer. N. Engl. J. Med. 349:419-421.
Esner, M., et al. 2006. Smooth muscle of the dorsal aorta shares a common clonal origin
with skeletal muscle of the myotome. Development. 133:737-749.
Tagliafico, E., et al. 2004. TGFbeta/BMP activate the smooth muscle/bone differentiation
programs in mesoangioblasts. J. Cell Sci. 117:4377-4388.
Cossu, G., Bianco, P. 2003. Mesoangioblasts — vascular progenitors for
extravascular mesodermal tissues. Curr. Opin. Genet. Dev. 13:537-542.